Dual inhibition of SUMOylation and MEK conquers MYC-expressing KRAS-mutant cancers by accumulating DNA damage.

BACKGROUND: KRAS mutations frequently occur in cancers, particularly pancreatic ductal adenocarcinoma, colorectal cancer, and non-small cell lung cancer. Although KRASG12C inhibitors have recently been approved, effective precision therapies have not yet been established for all KRAS-mutant cancers. Many treatments for KRAS-mutant cancers, including epigenome-targeted drugs, are currently under investigation. Small ubiquitin-like modifier (SUMO) proteins are a family of small proteins covalently attached to and detached from other proteins in cells via the processes called SUMOylation and de-SUMOylation. We assessed whether SUMOylation inhibition was effective in KRAS-mutant cancer cells. METHODS: The efficacy of the first-in-class SUMO-activating enzyme E inhibitor TAK-981 (subasumstat) was assessed in multiple human and mouse KRAS-mutated cancer cell lines. A gene expression assay using a TaqMan array was used to identify biomarkers of TAK-981 efficacy. The biological roles of SUMOylation inhibition and subsequent regulatory mechanisms were investigated using immunoblot analysis, immunofluorescence assays, and mouse models. RESULTS: We discovered that TAK-981 downregulated the expression of the currently undruggable MYC and effectively suppressed the growth of MYC-expressing KRAS-mutant cancers across different tissue types. Moreover, TAK-981-resistant cells were sensitized to SUMOylation inhibition via MYC-overexpression. TAK-981 induced proteasomal degradation of MYC by altering the balance between SUMOylation and ubiquitination and promoting the binding of MYC and Fbxw7, a key factor in the ubiquitin-proteasome system. The efficacy of TAK-981 monotherapy in immunocompetent and immunodeficient mouse models using a mouse-derived CMT167 cell line was significant but modest. Since MAPK inhibition of the KRAS downstream pathway is crucial in KRAS-mutant cancer, we expected that co-inhibition of SUMOylation and MEK might be a good option. Surprisingly, combination treatment with TAK-981 and trametinib dramatically induced apoptosis in multiple cell lines and gene-engineered mouse-derived organoids. Moreover, combination therapy resulted in long-term tumor regression in mouse models using cell lines of different tissue types. Finally, we revealed that combination therapy complementally inhibited Rad51 and BRCA1 and accumulated DNA damage. CONCLUSIONS: We found that MYC downregulation occurred via SUMOylation inhibition in KRAS-mutant cancer cells. Our findings indicate that dual inhibition of SUMOylation and MEK may be a promising treatment for MYC-expressing KRAS-mutant cancers by enhancing DNA damage accumulation.

Neutral selection and clonal expansion during the development of colon cancer metastasis.

Intratumor heterogeneity has been shown to play a role in the malignant progression of cancer. Although clonal evolution in primary cancer has been well studied, that in metastatic tumorigenesis is not fully understood. In this study, we established human colon cancer-derived organoids and investigated clonal dynamics during liver metastasis development by tracking barcode-labelled subclones. Long-term subclone co-cultures showed clonal drift, with a single subclone becoming dominant in the cell population. Interestingly, the selected subclones were not always the same, suggesting that clonal selection was not based on cell intrinsic properties. Furthermore, liver tumors developed by co-transplantation of organoid subclones into the immunodeficient mouse spleen showed a progressive drastic reduction in clonal diversity, and only one or two subclones predominated in the majority of large metastatic tumors. Importantly, selections were not limited to particular subclones but appeared to be random. A trend towards a reduction in clonal diversity was also found in liver metastases of multiple color-labeled organoids of mouse intestinal tumors. Based on these results, we propose a novel mechanism of metastasis development, i.e. a subclone population of the disseminated tumor cells in the liver is selected by neutral selection during colonization and constitutes large metastatic tumors.

Identification of uromodulin deposition in the stroma of perinephric fibromyxoid nephrogenic adenoma by mass spectrometry.

Nephrogenic adenoma (NA) is an epithelial lesion that usually occurs in the mucosa of the urinary tract. Rare cases of deep infiltrative or perinephric lesions have also been reported. Recently, NA with characteristic fibromyxoid stroma (fibromyxoid NA) has been proposed as a distinct variant. Although shedding of distal renal tubular cells due to urinary tract rupture has been postulated as the cause of NA in general, the mechanism underlying extraurinary presentation of NA and fibromyxoid stromal change in fibromyxoid NA remains unknown. In this study, we performed mass spectrometry (MS) analysis in a case of perinephric fibromyxoid NA of an 82-year-old man who underwent right nephroureterectomy for distal ureteral cancer. The patient had no prior history of urinary tract injury or radiation. Periodic acid-Schiff staining-positive eosinophilic structureless deposits in the stroma of fibromyxoid NA were microdissected and subjected to liquid chromatography/MS. The analysis revealed the presence of a substantial amount of uromodulin (Tamm-Horsfall protein). The presence of urinary content in the stroma of perinephric fibromyxoid NA suggests that urinary tract rupture and engraftment of renal tubular epithelial cells directly cause the lesion.

Gain-of-Function p53 Mutation Acts as a Genetic Switch for TGFß Signaling-Induced Epithelial-to-Mesenchymal Transition in Intestinal Tumors.

Signaling by TGFß family cytokines plays a tumor-suppressive role by inducing cell differentiation, while it promotes malignant progression through epithelial-to-mesenchymal transition (EMT). Identification of the mechanisms regulating the switch from tumor suppression to tumor promotion could identify strategies for cancer prevention and treatment. To identify the key genetic alterations that determine the outcome of TGFß signaling, we used mouse intestinal tumor-derived organoids carrying multiple driver mutations in various combinations to examine the relationship between genotypes and responses to the TGFß family cytokine activin A. KrasG12D mutation protected organoid cells from activin A-induced growth suppression by inhibiting p21 and p27 expression. Furthermore, Trp53R270H gain-of-function (GOF) mutation together with loss of wild-type Trp53 by loss of heterozygosity (LOH) promoted activin A-induced partial EMT with formation of multiple protrusions on the organoid surface, which was associated with increased metastatic incidence. Histologic analysis confirmed that tumor cells at the protrusions showed loss of apical-basal polarity and glandular structure. RNA sequencing analysis indicated that expression of Hmga2, encoding a cofactor of the SMAD complex that induces EMT transcription factors, was significantly upregulated in organoids with Trp53 GOF/LOH alterations. Importantly, loss of HMGA2 suppressed expression of Twist1 and blocked activin A-induced partial EMT and metastasis in Trp53 GOF/LOH organoids. These results indicate that TP53 GOF/LOH is a key genetic state that primes for TGFß family-induced partial EMT and malignant progression of colorectal cancer. Activin signaling may be an effective therapeutic target for colorectal cancer harboring TP53 GOF mutations. SIGNIFICANCE: KRAS and TP53 mutations shift activin-mediated signaling to overcome growth inhibition and promote partial EMT, identifying a subset of patients with colorectal cancer that could benefit from inhibition of TGFß signaling.

Genetic and nongenetic mechanisms for colorectal cancer evolution.

The stepwise accumulation of key driver mutations is responsible for the development and malignant progression of colorectal cancer in primary sites. Genetic mouse model studies have revealed combinations of driver gene mutations that induce phenotypic changes in tumors toward malignancy. However, cancer evolution is regulated by not only genetic alterations but also nongenetic mechanisms. For example, certain populations of metastatic cancer cells show a loss of malignant characteristics even after the accumulation of driver mutations, and such cells are eliminated in a negative selection manner. Furthermore, a polyclonal metastasis model has recently been proposed, in which cell clusters consisting of genetically heterogeneous cells break off from the primary site, disseminate to distant organs, and develop into heterogenous metastatic tumors. Such nongenetic mechanisms for malignant progression have been elucidated using genetically engineered mouse models as well as organoid transplantation experiments. In this review article, we discuss the role of genetic alterations in the malignant progression of primary intestinal tumors and nongenetic mechanisms for negative selection and polyclonal metastasis, which we learned from model studies.

In Vitro and In Vivo Models for Metastatic Intestinal Tumors Using Genotype-Defined Organoids.

It has been established that the accumulation of driver gene mutations causes malignant progression of colorectal cancer (CRC) through positive selection and clonal expansion, similar to Darwin's evolution. Following this multistep tumorigenesis concept, we previously showed the specific mutation patterns for each process of malignant progression, including submucosal invasion, epithelial mesenchymal transition (EMT), intravasation, and metastasis, using genetically engineered mouse and organoid models. However, we also found that certain populations of cancer-derived organoid cells lost malignant characteristics of metastatic ability, although driver mutations were not impaired, and such subpopulations were eliminated from the tumor tissues by negative selection. These organoid model studies have contributed to our understanding of the cancer evolution mechanism. We herein report the in vitro and in vivo experimental protocols to investigate the survival, growth, and metastatic ability of intestinal tumor-derived organoids. The model system will be useful for basic research as well as the development of clinical strategies.

Mapping Nanomechanical Properties of Basal Surfaces in Metastatic Intestinal 3D Living Organoids with High-Speed Scanning Ion Conductance Microscopy.

Studying mechanobiology is increasing of scientific interests in life science and nanotechnology since its impact on cell activities (e.g., adhesion, migration), physiology, and pathology. The role of apical surface (AS) and basal surface (BS) of cells played in mechanobiology is significant. The mechanical mapping and analysis of cells mainly focus on AS while little is known about BS. Here, high-speed scanning ion conductance microscope as a powerful tool is utilized to simultaneously reveal morphologies and local elastic modulus (E) of BS of genotype-defined metastatic intestinal organoids. A simple method is developed to prepare organoid samples allowing for long-term BS imaging. The multiple nano/microstructures, i.e., ridge-like, stress-fiber, and E distributions on BS are dynamically revealed. The statistic E analysis shows softness of BS derived from eight types of organoids following a ranking: malignant tumor cells > benign tumor cells > normal cells. Moreover, the correlation factor between morphology and E is demonstrated depending on cell types. This work as first example reveals the subcellular morphologies and E distributions of BS of cells. The results would provide a clue for correlating genotype of 3D cells to malignant phenotype reflected by E and offering a promising strategy for early-stage diagnosis of cancer.

Toll-like Receptor 9 Promotes Initiation of Gastric Tumorigenesis by Augmenting Inflammation and Cellular Proliferation.

BACKGROUND & AIMS: Gastric cancer (GC) is strongly linked with chronic gastritis after Helicobacter pylori infection. Toll-like receptors (TLRs) are key innate immune pathogenic sensors that mediate chronic inflammatory and oncogenic responses. Here, we investigated the role of TLR9 in the pathogenesis of GC, including Helicobacter infection. METHODS: TLR9 gene expression was profiled in gastric tissues from GC and gastritis patients and from the spontaneous gp130F/F GC mouse model and chronic H felis-infected wild-type (WT) mice. Gastric pathology was compared in gp130F/F and H felis infection models with or without genetic ablation of Tlr9. The impact of Tlr9 targeting on signaling cascades implicated in inflammation and tumorigenesis (eg, nuclear factor kappa B, extracellular signal-related kinase, and mitogen-activated protein kinase) was assessed in vivo. A direct growth-potentiating effect of TLR9 ligand stimulation on human GC cell lines and gp130F/F primary gastric epithelial cells was also evaluated. RESULTS: TLR9 expression was up-regulated in Helicobacter-infected gastric tissues from GC and gastritis patients and gp130F/F and H felis-infected WT mice. Tlr9 ablation suppressed initiation of tumorigenesis in gp130F/F:Tlr9-/- mice by abrogating gastric inflammation and cellular proliferation. Tlr9-/- mice were also protected against H felis-induced gastric inflammation and hyperplasia. The suppressed gastric pathology upon Tlr9 ablation in both mouse models associated with attenuated nuclear factor kappa B and, to a lesser extent, extracellular signal-related kinase, mitogen-activated protein kinase signaling. TLR9 ligand stimulation of human GC cells and gp130F/F GECs augmented their proliferation and viability. CONCLUSIONS: Our data reveal that TLR9 promotes the initiating stages of GC and facilitates Helicobacter-induced gastric inflammation and hyperplasia, thus providing in vivo evidence for TLR9 as a candidate therapeutic target in GC.

Genetic Alterations and Microenvironment that Drive Malignant Progression of Colorectal Cancer: Lessons from Mouse and Organoid Models.

Comprehensive genome analyses have identified frequently mutated genes in human colorectal cancers (CRC). These include APC, KRAS, SMAD4, TP53, and FBXW7. The biological functions of the respective gene products in cell proliferation and homeostasis have been intensively examined by in vitro experiments. However, how each gene mutation or combinations of specific mutations drive malignant progression of CRC in vivo has not been fully understood. Based on the genomic information, we generated mouse models that carry multiple mutations of CRC driver genes in various combinations, and we performed comprehensive histological analyses to link genetic alteration(s) and tumor phenotypes, including liver metastasis. In this review article, we summarize the phenotypes of the respective genetic models carrying major driver mutations and discuss a possible mechanism of mutations underlying malignant progression.

Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer.

Loss-of-function mutations in RNF43 induce activation of Wnt ligand-dependent Wnt/ß-catenin signaling through stabilization of the Frizzled receptor, which is often found in microsatellite instability (MSI)-type colorectal cancer (CRC) that develops from sessile serrated adenomas. However, the mechanism underlying how RNF43 mutations promote tumorigenesis remains poorly understood. In this study, we established nine human CRC-derived organoids and found that three organoid lines carried RNF43 frameshift mutations associated with MSI-high and BRAFV600E mutations, suggesting that these CRCs developed through the serrated pathway. RNF43 frameshift mutant organoids required both Wnt ligands and R-spondin for proliferation, indicating that suppression of ZNRF3 and retained RNF43 function by R-spondin are required to achieve an indispensable level of Wnt activation for tumorigenesis. However, active ß-catenin levels in RNF43-mutant organoids were lower than those in APC two-hit mutant CRC, suggesting a lower threshold for Wnt activation in CRC that developed through the serrated pathway. Interestingly, transplantation of RNF43-mutant organoids with intestinal myofibroblasts accelerated the ß-catenin nuclear accumulation and proliferation of xenograft tumors, indicating a key role of stromal cells in the promotion of the malignant phenotype of RNF43-mutant CRC cells. Sequencing of subcloned organoid cell-expressed transcripts revealed that two organoid lines carried monoallelic RNF43 cis-mutations, with two RNF43 frameshift mutations introduced in the same allele and the wild-type RNF43 allele remaining, while the other organoid line carried two-hit biallelic RNF43 trans-mutations. These results suggest that heterozygous RNF43 frameshift mutations contribute to CRC development via the serrated pathway; however, a second-hit RNF43 mutation may be advantageous in tumorigenesis compared with a single-hit mutation through further activation of Wnt signaling. Finally, treatment with the PORCN inhibitor significantly suppressed RNF43-mutant cell-derived PDX tumor development. These results suggest a novel mechanism underlying RNF43 mutation-associated CRC development and the therapeutic potential of Wnt ligand inhibition against RNF43-mutant CRC. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

STAT3-mediated upregulation of the AIM2 DNA sensor links innate immunity with cell migration to promote epithelial tumourigenesis.

OBJECTIVE: The absent in melanoma 2 (AIM2) cytosolic pattern recognition receptor and DNA sensor promotes the pathogenesis of autoimmune and chronic inflammatory diseases via caspase-1-containing inflammasome complexes. However, the role of AIM2 in cancer is ill-defined. DESIGN: The expression of AIM2 and its clinical significance was assessed in human gastric cancer (GC) patient cohorts. Genetic or therapeutic manipulation of AIM2 expression and activity was performed in the genetically engineered gp130 F/F spontaneous GC mouse model, as well as human GC cell line xenografts. The biological role and mechanism of action of AIM2 in gastric tumourigenesis, including its involvement in inflammasome activity and functional interaction with microtubule-associated end-binding protein 1 (EB1), was determined in vitro and in vivo. RESULTS: AIM2 expression is upregulated by interleukin-11 cytokine-mediated activation of the oncogenic latent transcription factor STAT3 in the tumour epithelium of GC mouse models and patients with GC. Genetic and therapeutic targeting of AIM2 in gp130 F/F mice suppressed tumourigenesis. Conversely, AIM2 overexpression augmented the tumour load of human GC cell line xenografts. The protumourigenic function of AIM2 was independent of inflammasome activity and inflammation. Rather, in vivo and in vitro AIM2 physically interacted with EB1 to promote epithelial cell migration and tumourigenesis. Furthermore, upregulated expression of AIM2 and EB1 in the tumour epithelium of patients with GC was independently associated with poor patient survival. CONCLUSION: AIM2 can play a driver role in epithelial carcinogenesis by linking cytokine-STAT3 signalling, innate immunity and epithelial cell migration, independent of inflammasome activation.

Nano-scale physical properties characteristic to metastatic intestinal cancer cells identified by high-speed scanning ion conductance microscope.

Recent genetic studies have indicated relationships between gene mutations and colon cancer phenotypes. However, how physical properties of tumor cells are changed by genetic alterations has not been elucidated. We examined genotype-defined mouse intestinal tumor-derived cells using a high-speed scanning ion conductance microscope (HS-SICM) that can obtain high-resolution live images of nano-scale topography and stiffness. The tumor cells used in this study carried mutations in Apc (A), Kras (K), Tgfbr2 (T), Trp53 (P), and Fbxw7 (F) in various combinations. Notably, high-metastatic cancer-derived cells carrying AKT mutations (AKT, AKTP, and AKTPF) showed specific ridge-like morphology with active membrane volume change, which was not found in low-metastatic and adenoma-derived cells. Furthermore, the membrane was significantly softer in the metastatic AKT-type cancer cells than other genotype cells. Importantly, a principal component analysis using RNAseq data showed similar distributions of expression profiles and physical properties, indicating a link between genetic alterations and physical properties. Finally, the malignant cell-specific physical properties were confirmed by an HS-SICM using human colon cancer-derived cells. These results indicate that the HS-SICM analysis is useful as a novel diagnostic strategy for predicting the metastatic ability of cancer cells.

Orchestration of myeloid-derived suppressor cells in the tumor microenvironment by ubiquitous cellular protein TCTP released by tumor cells.

One of most challenging issues in tumor immunology is a better understanding of the dynamics in the accumulation of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TIME), as this would lead to the development of new cancer therapeutics. Here, we show that translationally controlled tumor protein (TCTP) released by dying tumor cells is an immunomodulator crucial to full-blown MDSC accumulation in the TIME. We provide evidence that extracellular TCTP mediates recruitment of the polymorphonuclear MDSC (PMN-MDSC) population in the TIME via activation of Toll-like receptor-2. As further proof of principle, we show that inhibition of TCTP suppresses PMN-MDSC accumulation and tumor growth. In human cancers, we find an elevation of TCTP and an inverse correlation of TCTP gene dosage with antitumor immune signatures and clinical prognosis. This study reveals the hitherto poorly understood mechanism of the MDSC dynamics in the TIME, offering a new rationale for cancer immunotherapy.

FOXO3 is a latent tumor suppressor for FOXO3-positive and cytoplasmic-type gastric cancer cells.

FOXO3 is a member of the FOXO transcription factors thought to play a tumor-suppressor role in gastrointestinal cancer, while tumor-promoting function of FOXO3 has also been reported. These results suggest a context-dependent function of FOXO3 in tumor development. However, the relationship between the FOXO3 expression pattern and its role in tumorigenesis has not been elucidated. We examined the FOXO3 expression in 65 human primary gastric cancer and patient-derived xenograft tissues by immunohistochemistry and identified three subtypes according to subcellular localization: FOXO3-nuclear accumulated (FOXO3-Nuc), FOXO3-nuclear/cytoplasmic or cytoplasmic distributed (FOXO3-Cyt), and FOXO3-negative. In the FOXO3-Cyt gastric cancer cells, the expression of the constitutive active mutant FOXO3 (Act-ER FOXO3) induced the nuclear accumulation of FOXO3 and significantly suppressed colony formation and proliferation. The inhibition of the PI3K-AKT pathway by inhibitor treatment also suppressed the proliferation of FOXO3-Cyt gastric cancer cells, which was associated with the nuclear accumulation of endogenous FOXO3. Furthermore, the expression of Act-ER FOXO3 by an endogenous promoter significantly suppressed gastric tumorigenesis in Gan mice, a model of gastric cancer. Finally, treatment of FOXO3-Cyt human gastric cancer-derived organoids with an AKT inhibitor significantly suppressed the survival and proliferation. These results indicate that FOXO3 is a latent tumor suppressor for FOXO3-Cyt-type gastric cancer cells and that activation of the PI3K-AKT pathway protects this type of gastric cancer cell from FOXO3-mediated growth suppression via constitutive nuclear export. Thus, the inhibition of the PI3K-AKT pathway and nuclear translocation of endogenous FOXO3 may have therapeutic applications in the treatment of FOXO3-positive and cytoplasmic-type gastric cancer.

Malignant subclone drives metastasis of genetically and phenotypically heterogenous cell clusters through fibrotic niche generation.

A concept of polyclonal metastasis has recently been proposed, wherein tumor cell clusters break off from the primary site and are disseminated. However, the involvement of driver mutations in such polyclonal mechanism is not fully understood. Here, we show that non-metastatic AP cells metastasize to the liver with metastatic AKTP cells after co-transplantation to the spleen. Furthermore, AKTP cell depletion after the development of metastases results in the continuous proliferation of the remaining AP cells, indicating a role of AKTP cells in the early step of polyclonal metastasis. Importantly, AKTP cells, but not AP cells, induce fibrotic niche generation when arrested in the sinusoid, and such fibrotic microenvironment promotes the colonization of AP cells. These results indicate that non-metastatic cells can metastasize via the polyclonal metastasis mechanism using the fibrotic niche induced by malignant cells. Thus, targeting the fibrotic niche is an effective strategy for halting polyclonal metastasis.

Autophagy regulates levels of tumor suppressor enzyme protein phosphatase 6.

Protein phosphatase 6 (PP6) is an essential serine/threonine protein phosphatase that acts as an important tumor suppressor. However, increased protein levels of PP6 have been observed in some cancer types, and they correlate with poor prognosis in glioblastoma. This raises a question about how PP6 protein levels are regulated in normal and transformed cells. In this study, we show that PP6 protein levels increase in response to pharmacologic and genetic inhibition of autophagy. PP6 associates with autophagic adaptor protein p62/SQSTM1 and is degraded in a p62-dependent manner. Accordingly, protein levels of PP6 and p62 fluctuate in concert under different physiological and pathophysiological conditions. Our data reveal that PP6 is regulated by p62-dependent autophagy and suggest that accumulation of PP6 protein in tumor tissues is caused at least partially by deficiency in autophagy.

Loss of wild-type p53 promotes mutant p53-driven metastasis through acquisition of survival and tumor-initiating properties.

Missense-type mutant p53 plays a tumor-promoting role through gain-of-function (GOF) mechanism. In addition, the loss of wild-type TP53 through loss of heterozygosity (LOH) is widely found in cancer cells. However, malignant progression induced by cooperation of TP53 GOF mutation and LOH remains poorly understood. Here, we show that mouse intestinal tumors carrying Trp53 GOF mutation with LOH (AKTPM/LOH) are enriched in metastatic lesions when heterozygous Trp53 mutant cells (AKTP+/M) are transplanted. We show that Trp53 LOH is required for dormant cell survival and clonal expansion of cancer cells. Moreover, AKTPM/LOH cells show an increased in vivo tumor-initiating ability compared with AKTPNull and AKTP+/M cells. RNAseq analyses reveal that inflammatory and growth factor/MAPK pathways are specifically activated in AKTPM/LOH cells, while the stem cell signature is upregulated in both AKTPM/LOH and AKTPNull cells. These results indicate that TP53/Trp53 LOH promotes TP53/Trp53 GOF mutation-driven metastasis through the activation of distinct pathway combination.

Inflammatory and mitogenic signals drive interleukin 23 subunit alpha (IL23A) secretion independent of IL12B in intestinal epithelial cells.

The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11-7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.

Variant and Invariant Spatiotemporal Structures in Kinematic Coordination to Regulate Speed During Walking and Running.

Humans walk, run, and change their speed in accordance with circumstances. These gaits are rhythmic motions generated by multi-articulated movements, which have specific spatiotemporal patterns. The kinematic characteristics depend on the gait and speed. In this study, we focused on the kinematic coordination of locomotor behavior to clarify the underlying mechanism for the effect of speed on the spatiotemporal kinematic patterns for each gait. In particular, we used seven elevation angles for the whole-body motion and separated the measured data into different phases depending on the foot-contact condition, that is, single-stance phase, double-stance phase, and flight phase, which have different physical constraints during locomotion. We extracted the spatiotemporal kinematic coordination patterns with singular value decomposition and investigated the effect of speed on the coordination patterns. Our results showed that most of the whole-body motion could be explained by only two sets of temporal and spatial coordination patterns in each phase. Furthermore, the temporal coordination patterns were invariant for different speeds, while the spatial coordination patterns varied. These findings will improve our understanding of human adaptation mechanisms to tune locomotor behavior for changing speed.

CRISPR-Cas9-mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beauty transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-ß (TGF-ß) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.